Sybr green precision mastermix

11,500 HDFcm⁻² were plated into dishes, reaching 80% confluency within 48 hours, and then serum-starved overnight, before treatment in SFM alone or supplemented with Salbutamol or Formoterol (10 μM). At 6 hours post treatment, total RNA was isolated (Nucleospin RNA II kit, Macherey Nagel, Duren, Germany) with DNase treatment. A measure of 1 μg of total RNA was reverse-transcribed using oligo-dT (NanoScript RT kit, Primer Design, Southampton, UK). Duplicate PCR reactions were performed with 10 ng of cDNA, 300 nM primers, and SYBR green Precision MasterMix (Primer Design) on a LightCycler 480 (Roche, Welwyn, UK), followed by a melting curve. Type I collagenA1, CCN2, FGF2, SMA, and TGF-β1 primers were purchased from Primer Design, whereas primers for β2AR and FN EDA were custom-designed using NCBI Primer Blast and synthesized by Sigma-Aldrich (Gillingham, UK). Reference genes CYC1 and UBE2D2 were selected using a geNorm kit (Primer Design) and qBase+ software (Biogazelle; Zwijnaarde, Belgium). Using the average Cq of CYC1 and UBE2D2 for normalization, mRNA relative expression level was calculated by the 2^−ΔΔCq method.